According to researchers at the National Cancer Institute (NCI), part of the National Institutes of Health (NIH), the expression of a protein that promotes cell death is reduced in human cancer cells but increased in cells of the normal connective tissue, or stroma, that surrounds a tumor. Elevated expression of this protein, CLIC4, in the stroma is predictive of cancer progression and associated with poor prognosis. Results of this study appear in the January 1, 2007, issue of Clinical Cancer Research*.

"It is particularly important that we understand the molecular and cellular changes that occur during tumor development in the tumor microenvironment," said NCI Director John Niederhuber, M.D. "This new knowledge is crucial for the discovery of future therapeutic targets for cancer."

Members of the CLIC (chloride intracellular channel) family of proteins are found in various organelles, or compartments, in the cells of many different types of tissue throughout the body. Anchored in membranes, these proteins form channels that allow chloride ions to pass into and out of membrane-bound organelles such as mitochondria, which are specialized structures that are responsible for energy production in the cell. The regulation of chloride ion flow is instrumental in maintaining normal cell volume, electrolyte balance, and a neutral electrostatic charge, all of which are vital for a healthy cell environment.

CLIC proteins also move into and out of the nucleus, where genetic material is stored. In response to stress or DNA damage, CLIC4 protein molecules that are not yet anchored in a cell membrane relocate to the nucleus. This relocation not only activates the cell death pathway but also accelerates it, which is often necessary to prevent uncontrolled growth and the division of damaged cells.

In this study, NCI researchers led by Stuart H. Yuspa, M.D., lab chief of the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, and his colleague, Kwang S. Suh, Ph.D., used tissue array analysis, a technique that detects specific proteins in multiple tumor samples at the same time, to compare CLIC4 levels in normal and tumor tissues derived from patients. They showed that the CLIC4 protein is absent in the nucleus of tumor cells and reduced in tumor tissue. The decline in CLIC4 protein in tumor cells and a coincident increase in CLIC4 production in cells of the surrounding stroma were common events during cancer progression in about 80 percent of all major cancer types tested, including breast, lung, prostate, and several others. In addition, declines in CLIC4 in the tumor and increases in the stroma correlated with tumor progression and severity of the disease.

In their next step, the researchers used a highly malignant human breast cancer cell line to establish tumors in mice. Then, they injected genetically engineered viruses that were capable of producing the CLIC4 protein into the established tumors and found that tumor growth was inhibited. In addition, when the scientists spurred the stromal cells surrounding similarestablished tumors to overproduce CLIC4, they observed enhanced tumor growth, suggesting an interaction or "crosstalk" between the tumors and the surrounding cells.

"At the moment, we're not sure what happens in tumor cells to silence production of the CLIC4 protein, since the gene is still intact," said Yuspa. "Hopefully, once we have more information, targeting CLIC4 in the tumor, the stroma, or both, will provide new opportunities for inhibiting tumor growth."

Understanding how CLIC4 altered the stromal environment to support tumor growth was another part of the researchers' study. They found that increased production of CLIC4 protein in stromal cells coincided with the conversion of fibroblasts, the connective tissue cells that make up most of the tumor-surrounding environment, to myofibroblasts, a different cell type. This change is marked by increased levels of a protein involved in cell structure and movement, α-smooth muscle actin (αSMA). In the laboratory, myofibroblasts contribute to tumor growth and spread by secreting enzymes and promoting the development of new blood vessels. The presence of myofibroblasts in tumor stroma is a very poor prognostic sign.

By growing fibroblasts and human tumor cells together in the laboratory in such a way that the tumor cells grew as a small colony with fibroblasts surrounding them, Yuspa and his colleagues found that the fibroblasts in the regions immediately adjacent to the tumor cells increased their production of CLIC4 and αSMA, but fibroblasts distant from the tumor cells did not. This observation indicates that tumor cells somehow stimulate fibroblasts to pump up production of these two proteins and suggests that crosstalk between tumors and healthy tissue is essential for tumor growth.

"Producing factors to influence surrounding cells is a devilish way for tumors to enhance their own growth," said Yuspa. "We are currently working to further characterize the signaling between a tumor and its stroma and to understand the relationship between CLIC4 and αSMA."

For more information about cancer, please visit the NCI Web site at http://www.cancer.gov, or call NCI's Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

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